The present application relates to a base sequence analysis method, a base sequence analysis apparatus, and a base sequence analysis program. More specifically, the present application relates to a base sequence analysis method and the like that includes a turbidity measurement procedure and a melting curve analysis procedure.
The temperature at which a double-stranded nucleic acid chain dissociates (melts) into two single-stranded nucleic acids is called the melting temperature (Tm value). In melting curve analysis, change in a signal detection amount with respect to change in temperature is measured by detecting a signal that changes due to the melting that occurs when a double-stranded nucleic acid chain is gradually heated and melts into two single-stranded nucleic acids. The melting temperature can be determined from a melting curve obtained by plotting the changes in signal detection amount with respect to change in temperature. Since the melting temperature reflects the homology between the two single-stranded nucleic acids, the homology between the two single-stranded nucleic acids can be determined based on the melting temperature. Consequently, melting curve analysis is utilized for analyzing nucleic acids, such as verification of the specificity of a nucleic acid amplification reaction and genotype determination of single nucleotide polymorphisms (SNPs) and the like.
A fluorescent substance is commonly used to detect the melting of a double-stranded nucleic acid chain. For example, by fluorescently labeling a nucleic acid probe capable of forming a double-stranded chain with a target nucleic acid chain, dissociation of the nucleic acid chain can be detected based on the emission or extinguishing of light by the fluorescent label when the target nucleic acid chain and the nucleic acid probe dissociate. Further, change from a double strand to a single strand can be detected using an intercalator that produces fluorescent light by intercalating into a double-stranded nucleic acid.
Fluorescent substances are not limited to melting curve analysis, they are also widely used in detection and quantification of an amplified nucleic acid chain and other such nucleic acid analysis. On the other hand, for amplified nucleic acid chain detection, methods that detect the turbidity of a sample without using a fluorescent substance are also employed. In an extension reaction using a DNA synthesis enzyme, pyrophosphoric acid produced as a byproduct binds with magnesium ions in the reaction solution, thereby forming magnesium pyrophosphate. If the amount of produced magnesium pyrophosphate exceeds the soluble level in the sample, magnesium pyrophosphate precipitates, and the reaction solution turns cloudy. By measuring the level of cloudiness, detection and quantification of an amplified nucleic acid chain can be carried out.
Accordingly, for example, JP-A-2012-34617 discloses a “nucleic acid amplification reaction apparatus including a first light source configured to output light that excites a fluorescent substance, a second light source configured to output light in a wavelength region that matches a wavelength region of fluorescent light produced from the fluorescent substance, and a control unit configured to emit light by switching between the first light source and the second light source, wherein an amount of light produced from turbid matter formed as the amplification reaction progresses and an intensity of fluorescent light produced from the fluorescent substance excited by the light as the amplification reaction progresses can be detected by passing the light from the first light source and the light from the second light source along an optical path where both beams of light are made to overlap by a light guidance member, and irradiating on a reaction area serving as a reaction site of a nucleic acid amplification reaction.